Oxamicetin and process for its production

ABSTRACT

The specification discloses a new chemical substance designated as oxamicetin and a process for preparing the substance by cultivation of a strain of Arthrobacter named Arthrobacter oxamicetus. Oxamicetus has been shown to have antibiotic activity against both Gram-positive and Gram-negative organisms.

United States Patent Kawaguchi et al.

[ Aug. 26, 1975 OXAMICETIN AND PROCESS FOR ITS PRODUCTION Inventors: Hiroshi Kawaguchi, Tokyo;

Masataka Konishi, Yokohama; Koji Tomita, Kawasaki, all of Japan Assignee: Bristol-Myers Company, New York,

Filed: Feb. 28, 1974 Appl. No.1 446,846

Related [1.8. Application Data Continuation of Sen No 265.402, June 22, 1972, Pat. No. 3,843,449.

US. CL... 260/2115 AB; 195/96; 260/210 AB;

260/211 R; 260/2115 R Int. Cl. C07H 19/06 Field of Search... 260/210 AB, 211.5 R, 210 R References Cited OTHER PUBLICATIONS Stanek et al., The Monosaccharides," Academic Press, Inc., New York, N.Y., 1963, pp. 471-472.

Primary Examiner-Johnnie R. Brown Attorney, Agent, or Firm.lames Magee, Jr.

[57] ABSTRACT 1 Claim, No Drawings 1 2 OXAMICETIN AND PROCESS FOR ITS neutral and alkaline water, but practically insoluble in PRODUCTION acetone, ethyl acetate, ether and other common or- This is a continuation of application Ser. No. ganic solvents. 265,402, filed June 22, 1972, now US. Pat. No. Crystalline oxamicetin hydrochloride melts at 205 3,843,449. 5 C. to 210 C. with decomposition. Potentiometric titration of the hydrochloride in 50% aqueous ethanol BACKGROUND OF THE INVENTION showed the presence of three titrable groups in the This invention is directed to a heretofore unknown molecule; one acidic group with a pKa of 1 1.2 and two chemical substance, hereafter referred to as oxamicebasic groups with the same pKa' value of 6.70, the titratin, and to a process for preparing oxamicetin by fer- [0 tion equivalent being 748. Analysis corresponds to the mentation of a new species of the Genus Arthrobacter emperical formula: C H N O ZHCLZH O (MW designated as Arthrobacter oxamicetus. This organism 743.6).

was isolated from a soil sample collected at Kominato, Calcd.: C 46.84, H 6.51, N 11.30.

Chiba. Japan. A culture of the organism has been de Found: C 47.22, H 6.58, N l 1.29.

posited with the American Type Culture Collection, Optical rotation of oxamicetin hydrochloride is:

Rockville, Maryland, as ATCC 21788. [01],, =+66 (c 0.4, water). The ultraviolet absorption Oxarnicetin, which has the structure, spectrum is shown in FIG. 1. The absorption maxima J HO l l\\ 0H CH NIL,

is a basic antibiotic which can be crystallized as its hyare observed at 305 my (6 1 31,700) in water, 316 mp drochloride hydrate. Elemental analysis and titration (e 26,600) in 0.1N HCl and 322 my. (6 21,900) in data have been found to be consistent with a molecular 0.1N NaOH. The infrared spectrum of oxamicetin hyformula of cggHqgNqo n for the free base. This formula drochloride in KBr pellet is shown in FIG. 2, which sugcontains one oxygen atom in excess of the amicetin gested a close resemblance to that of amicetin.

molecule reported by De Boer et al. in J. Am. Chem. Soc. 75:499 1953). Oxamicetin contains 3- hydroxyamicetose (D-ehromose C or olivose) in place TLC detected b'o'autography Subnhs or by of amicetose found in the structure of amicetin. spraymg anthrone reagent:

The compound described above is produced by culti- 40 (D BUOH ACOIT" HZO 3: 1 I (EtOH a vating an oxamicetin-producing strain of Arthrobacter NH4OH Alumma TLC develope with in an aqueous carbohydrate medium containing a 80% methanol was found suitable to differentiate oxatrogenous nutrient under submerged aerobic condimlcem (Rf: 0'54) from am'cetm (Rf I oxamicetin gives the following Rf values on silicagel tions. The antibiotic compound can be recovered from The minimum inhibitory concentration (Mlc) f the fermentation broth y usual methods including amicetin was determined by the serial agar dilution organic Solvent extraction method. The results are shown in Table l, below, along The infrared Spectrum Show in 1 of the with those obtained with amicetin which was used as a tached drawing shows absorptions due to hydroxy reference,

group (3300 cm), amide carbonyls 1690, 1640) and Conjugated C=C bond (1600 1 The compound oxamicet n exhibits slm|lar antibacterial spectrum to gave positive ninhydrin, Dragendorff and anthrone rea f f somwhat l acnve than actions but is negative to Fehling and Tollens reagents. almcetm against Grarrl'negatwe i f but i indicating the absence of reactive reducing sugar tive than the latter against Gram-positive and acid-fast eties. The NMR spectrum of oxamicetin hydrochloride bactena in deuterium oxide shows three CcH1h two The in vivo activity of oxamicetin was assessed by the blets Camel-ed at 5144 f and one singlet experimental infection in mice against two pathogenic at 8 and 9 dlmefllylammo group at 8 309 bacteria, S. aureus Smith and E. coli NlHJ. Mice were The charactensuc two vmyl protons appeajred as inoculated intraperitoneally with a 100xLD dose of doublets at 5 and ppm and four ammauc the pathogen and oxamicetin was administered subcutons as an AB quartet centered at 6 taneously just after the bacterial challenge. Oxamicetin tion two anomeric protons are observed at 5 protected mice from the infection, giving subcutaneous (doublet, J 3-5HZ) and 570 (mad doublet J co of mg./kg. against s. aureus Smith and I05 Hz). These NMR data reflect the close structural "lg/kg. against E- co No in vivo activity was similarity of this antibiotic to amicetin. The chemical 886 when the antibiotic was given orally shift of the signal clearly distinquishes oxamicetin from amicetin. The acute toxicity of oxamicetin was determined in oxamicetin-free base is readily soluble in acidic wamice, the intravenous LD being 200 mg./kg. it was ter, methanol, ethanol and n-butanol, slightly soluble in nontoxic at 400 mg./kg. when given subcutaneously.

TABLE I MlC (meg/ml.) TEST ORGANISMS TEST OXAMlCE- AMICETIN MEDIUM TIN Escherichia coli NIH] A 25 50 Escherichia coli .luhl A 100 100 Escherichia coli A1 5169 A 6.3 6.3 Escherichia coli A20365 A 6.3 12.5 Klebsiella pneumoniae D1 1 A 50 100 Proteus vulgaris A9436 A 50 50 Proteus vulgaris A9526 A 25 25 Proteus morganii A2003] A 50 100 Proteus mirabilis A9554 A 100 100 Shigella flexneri A9684 A 100 100 Shigella sonnei Yale A 50 100 Salmonella enteritidis A9531 A 3.1 3.1 Salmonella typhosa Yale A 50 100 Pseudomonus aeruginosa D15 A 100 100 Staphylococcus aureus Smith A 12.5 6.3 Staphylococcus aureus 93 A 12.5 6.3 Staphylococcus aureus Russell A 12.5 6.3 Staphylococcus aureus Terajima A 1.6 0.8 Streptococcus pyogenes 5-23 B 6.3 3.1 Streptococcus pyogenes Dick B 100 50 Streptococcus pyogenes Digonnet B 25 6.3 Diplococcus pneumoniae Type 2 B 25 3.1 Sarcina lutea PCI A 1.6 1.6 Micrococcus flavus A 1.6 3.1 Bacillus subtilis PCl-219 A 6.3 12.5 Bacillus anthracis 1 A 12.5 Mycobacterium 607 C 12.5 3.1 Mycobacterium 607 C 12.5 3.1 Mycobacterium 607 C 3.1 1.6 Mycobacterium phlei C 3.1 0.8 Mycobacterium ranae C 12.5 3.1

A: neutrient agar. B: blood agar.

ammonium citrate. 0.001"; MgSO,. 1.5; agar.

The organism used in the production of oxamicetin was found to grow well at 20 C. to C. on agar slants. Such slants were then used to prepare seed cultures by innoculation of a liquid vegetative medium containing about 2% glycerol, about 1% Pharmamedia, about 1% corn steep liquor, about 0.3% ammonium sulfate, about 0.4% calcium carbonate and about 0.003% hydrated zinc sulfate (zuSO .7l-l O). The seed culture was incubated at about 28 C. for 2 days on a rotary shaker running at 250 rpm. About 2 ml. of the seed culture was used to start a fermentation by transfer to 100 ml. of fermentation medium in a half liter Erlenmeyer flask. The composition of the fermentation medium was the same as that of the seed culture. Progress of the fermentation was followed by paper disc-agar difi'usion assay using Bacillus subtilis PC1219 as a test organism. Antibiotic production in shaken flasks has been observed on the second day giving a potency of about 180 mcg. per m1. at a pH of about 6.9. A maximum potency of about 250 mcg. per ml. was reached on the third day (pH 7.7).

Oxamicetin has been produced by fermentation in 10 liter jar fermentors and in 100 liter pilot plant tanks using the above-described medium. In general, peak potency of about 200 mcg. per ml. was obtained in about 50 to 60 hours.

The fermentation broth (50L) was filtered at pH 3, and the filtrate was extracted at pH 8.2 to 8.4 with about one-third volume of n-butanol (15L). The extract was stirred with 5L of acidic water. the pH being adjusted to 2.0 by dilute hydrochloric acid. The active aqueous extract was then made alkaline (pH 8.2 to 8.4) and extracted again with 3L of n-butanol. The hutanol extract was washed by water, concentrated in vacuo and then lyophilized to give about 6 g. of yellowish powder (potency: ca 500 meg/mg). Alumina thinlayer chromatography (TLC) developed by methanol indicated a presence of slower moving impurity in the crude preparation.

Further purification was effectively accomplished by alumina column chromatography. Crude oxamicetin (3 g.) was dissolved in 50 ml. of 0.5N methanolic hydrogen chloride, and the solution was applied on a column of acid-treated alumina, the column being eluted fractionally by methanol. The active fractions were combined and concentrated in vacuo to dryness to afford 1.2 g. of oxamicetin hydrochloride with to purity. The solid was dissolved in a small amount of water containing a drop of hydrochloric acid and the solution was added with acetone to a cloudy point. Overnight storage of the solution in the cold gave colorless needle-like crystals of oxamicetin hydrochloride.

Arthrobacter oxamicetus, ATCC 21788, is a nonsporulating, non-motile bacterium and the cells show morphological changes during the growth, and these features are characteristic of the Family Corynebacteriaceae.

For the following characterization of this organism, the procedures described by Skerman. Wileylnterscience, 1969; Gibbs et al.. The Society for Applied Bacteriology. Technical Series 1, 1966; Gibbs et al., The Society for Applied Bacteriology, Technical Series 2. 1968; were followed. For the taxonomic identification of the organism, the descriptions by Conn et al., J. Bacteriol. 54: 291-303 (1947); Nigel et al., J. Bacteriol. 90(4): 921927 1965); and Cummins et al., Nature 184: 831-832 (1959) were used.

Morphological Characteristics A conspicuous characteristic of this organism is a pleomorphism of the cells in size and shape during the course of growth (Plates 1 & 2). Young cells 12 to 24 hours) are Gram-variable (generally positive) rods of several lengths with irregular shapes, being straight, bent, curved, filamentous, and occasionally with a rudimentary branch. These rods develop, after 32 hours or later, into Gram-positive cocci of various forms. They may be single, paired or chained cocci, and the chains are straight, curved and zigzag.

Size of the cells ranges 0.5 to 0.8 X 0.6 to 4.0 microns. The organism is non-sporulating and non-motile and the acid-fast stain reaction (Ziehl-Neelsen) is negative.

Arrhrobacter oxamicetus developed into two forms on the YGA-agar plate (glucose asparagine agar 0.05% yeast extract), a compact type and a diffuse type.

Compact type of YGA agar colonies: Small, 0.3 to 1.0 mm. in diameter (3rd days). Compact, raised, convex and circular. Smooth, rigid and dull. Opaque, whitish to cream-colored, later light-pink orange. No soluble pigment.

Diffuse type of YGA agar colonies: Larger than compact type, 0.7 to 2.0 mm. (3rd days). Difi'used, circular and less raised. Smooth, soft and glistening. Opaque, cream-colored, later light-pink orange. No soluble pigment.

YGA agar slant: Abundant growth. Smooth, soft, opaque, cream-colored, later light-pink orange. Not viscous. No soluble pigment.

Nutrient agar slant: Abundant growth. Aged cell not colored. Others same as YGA agar.

Nutrient broth: Light turbidity with sediment. No surface growth. No pigmentation.

Growth temperature: Scant or no growth at 37 C.

0 Restricted growth at 32 C. to 35 C. Good growth at 20 C. to C.

Oxygen demand: Obligately aerobic. NaCl broth: No growth at 8%-NaCl. Restricted growth at 4 to 6% NaCl. Good growth at 0.5 to 3% NaCl.

The results of the physiological reactions and the carbohydrate utilization test on strain ATCC 21788 are 5 shown in Table II and Table III, below, respectively.

TABLE II Test Response Method and Medium Employed Survival test at 72 C. Decomposition of cellulose Utilization of ammonium salts as a sole nitrogen source Utilization of citrate as Not survived l0 minutes in milk a sole carbon source Pigment from nicotine Starch hydrolysis Nitrite from nitrate Gelatin liquefaction Milk peptonizalion Milk coagulation Change of pH in milk Indole production VogesrProskauer reaction ",5 production from cysteine and thiosulfate Gas from carbohydrate Urease reaction Catalase reaction Oxidase reaction Oxidative vs. fermentative metabolism of Negative Inorganic salts plus 0.05% yeast extract Positive Inorganic salts plus I% sugar Positive Simmons citrate agar Negative Nicotine agar (Sguros, (no growth) I955) Negative Hayward's starch agar Negative Peptone broth plus 0.1% I ZNO Negative Peptone broth plus 25% gelatin (Skerman, I967) Negative Incubation at 22 C.

for 3 weeks Positive Incubation at 22 C. (Slowly coagfor 3 weeks ulated) Slightly Incubation at 22" C. for acidified three weeks. Negative Peptone broth (Kovacs' reagent) Negative Peptone broth plus I% glucose Negative Skerman s I967) method,

and lead acetate agar Negative Glucose, sucrose and mannitol as carbohydrate Positive Christensen s urea medium Positive Hydrogen-peroxide solution Negative Reaction of p-aminodimethylaniline oxalate (Kovacs oxidase reagent) Oxidative Sucrose and mannitol as or no acid carbohydrate (Hugh 8L carbohydrate Leifson, 1953) TABLE III CARBOHYDRATE UTILIZATION OF STRAIN ATCC 2l788 Glycerol Maltose L-Arahinose Raftinosc D-Xylose lnositol Rhamnosc D-Mannitol D-Fructosc D Sorbitol D-Galactnse Dulcitol D-Glucosc Starch A D-Mannose Cellulose Sucrose Inuline Lactose Salicinc Taxonomy in the 7th Edition of Bergey's Manual of Determinative Bacteriology" (1957), six genera of Family Corynebacteriaceae are described (pages 578-612), among which three genera of Corynebacterium, Listeria and Erysipelothrix are clearly differentiated from Arthrobacter oxamicetus. Furthermore, Genus Microbacterium is thermoduric (at 72 C.) and Genus Cellulomonas decomposes cellulose, and therefore these two genera are also different from the present organism. Genus Arthrobacter is a soil organism, does not decompose cellulose, and the cells are pleomorphic and generally non-motile. These properties accord with those of Arthrobacter oxamicetus and, therefore, place it in the Genus Arthrobacter.

Smeath et al., lnterntl. .1. System. Bacterio]. 16(1): 1-7 (1966), described five additional species of Arthrobacter as the type strains. They are A. atrocyaneus, A. duodecadis, A. flavescens, A. nicotianae and A. ramosus. A. oxamicetus differs from A. flavescens and A.

N-- ca,

duodecadis in the requirement of vitamin 8, and terregens factor, from A. arrocyaneus in the blue pigmentation, optimum growth temperature, urease activity and starch hydrolysis, from A. nicotianae in the pigmentation, and from A. ramosus in the gelatin liquefaction and chromogenicity.

A. oxamicetus was further compared with five Arthrobacter species including those appeared in the recent literatures. They are A. cryslallopoietes, A. polychromogenes, A. viscosus, A. marinas and A. sp. NRRL- B338l (erythromycinproducer) US. Pat. No. 3,551,294.

A. viscosus shares several common features with A. oxamicetus but differs from the latter in the lack of pigment on sugar media, the peptonization but no coagulation of milk, the nitrate reduction and the production of viscous polysaccharide.

Oxamicetin has properties which make it useful as an antibacterial agent, nutritional supplement in animal feeds, therapeutic agent in poultry and animals, including man. This compound is especially valuable in the treatment of infectious diseases caused by either Grampositive or Gram-negative bacteria. Antibacterial compositions containing oxamicetin can be prepared by the usual pharmaceutical techniques and administered in accordance with accepted and standard medical practice.

What is claimed is:

l. The compound oxamicetin characterized by the structure 

1. THE COMPOUND OXAMICETIN CHARACTERIZED BY THE STRUCTURE 